Article pubs.acs.org/JPCC
Oligonucleotide Functionalization of Hollow Triangular Gold Silver Alloy Nanoboxes Gemma L. Keegan,† Damian Aherne,† Eric Defrancq,‡ Yurii K. Gun’ko,† and John M. Kelly*,† †
School of Chemistry, Trinity College Dublin, Dublin 2, Ireland Département de Chimie Moléculaire, UMR CNRS 5250, Université Joseph Fourier, BP 53, 38041 Grenoble Cedex 9, France
‡
S Supporting Information *
ABSTRACT: Triangular AuAg nanoboxes functionalized with short DNA molecules (oligodeoxynucleotides, ODNs) have been prepared and their properties compared to those formed from spherical gold particles. The nanoboxes, produced from triangular silver nanoplates (TSNPs) by reduction of AuCl4− with ascorbic acid, have been further characterized by EDS mapping and HAADF-STEM measurements. Both AuAg nanoboxes and gold spherical nanoparticles were functionalized with thiol-terminated single-stranded ODNs or their complementary ODN sequences. When the complementary AuAg nanobox−ODN conjugates were combined they were observed to form networks, with the localized surface plasmon resonance (LSPR) band undergoing a red shift and a significant dampening. This assembly process was reversible upon heating and the systems showed sharp melting transitions, which could be monitored at wavelengths throughout the visible and near-IR. Finally, assemblies of the triangular nanoboxes and spherical nanoparticles have been generated.
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etching by salt.38 Depending on the amount of gold available for reaction a range of structures can be prepared from the TSNPs. Of particular interest are the hollow nanoboxes formed when the molar ratio of Ag:Au is approximately 1:3. As well as their unusual morphology these particles are potentially useful as the position of the LSPR band is tunable by altering the thickness of the wall of the particles, which allows the LSPR band to be tuned without drastically altering the nanoparticle shape, size, and aspect ratio.39 There have been many reports on the functionalization of metal nanoparticles with oligodeoxynucleotides (ODNs) following the initial reports with spherical gold in 1996 and they have been used to form assemblies and in the area of detection.40,41 More recently this has been extended to anisotropic nanoparticles,42−44 with just one account of the direct functionalization of TSNPs where however the particle morphology was not fully maintained.45 Xue et al. reported the ODN functionalization of silica coated triangular silver nanoplates, but the thinnest complete silica layer possible was shown to be 15 nm.46 This may limit the usefulness of the nanoplates, as phenomena such as metal enhanced fluorescence (MEF) have been shown to have a strong dependence on the distance separating the probe molecule from the nanoparticle surface. Also the LSPR of the nanoplates is much less sensitive to changes in their local environment outside the silica coating. While some related hollow metal nanostructures have been functionalized with antibodies and hormones,47−49 to the best
INTRODUCTION Metal nanoparticles have been the subject of intense investigation in part due to the potential of their unique optical properties, which are a consequence of their localized surface plasmon resonance (LSPR).1−7 In the case of anisotropic particles, the LSPR can result in large enhancements of the local electric field surrounding the particle and this has been implicated in observed phenomena such as surface enhanced Raman scattering (SERS)8−11 and metal enhanced fluorescence (MEF).12−16 Triangular silver nanoplates (TSNPs) are particularly attractive as they can be readily formed and their size easily controlled. A number of methods for their synthesis have been reported,11,17−24 a particularly convenient method being the room temperature, seed-catalyzed ascorbic acid reduction of silver ions.25 This method allows one to prepare samples with edge lengths ranging from less than 20 nm up to greater than 100 nm, which causes the LSPR to shift from about 480 nm to greater than 1100 nm, with the color changing from red through blue to almost colorless, as the size increases. While silver nanoparticles have several synthetic advantages, as well as higher extinction coefficients than their gold counterparts,26 there are some disadvantages, primarily the instability in the presence of halide anions.3,27−35 This may cause difficulties during functionalization as well as for practical applications, e.g., in biological media. Additionally attachment of functionality through thiols is more problematical for silver than for gold because of the weaker Ag−S bonds.36,37 Coating the TSNPs with gold may provide a possible route to overcoming these difficulties, as it has already been shown that treating the TSNPs with gold stabilizes them against © 2012 American Chemical Society
Received: September 23, 2012 Revised: November 26, 2012 Published: December 4, 2012 669
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step of the automated synthesis by using an extended coupling time. ODNs were cleaved from the CPG solid support by treatment with 28% aqueous ammonia solution for 1 h followed by 16 h at 55 °C for nucleobase deprotection. The ODNs were purified by semipreparative reverse phase HPLC (RP-HPLC). RP-HPLC analysis was performed on a Gilson HPLC system with a dual wavelength detector using a μ-Bondapak C-18 column (Macherey-Nagel Nucleosil, 250 × 10 mm, 7 μm) with a solvent system of; solvent A (50 mM TEAAc buffer containing 5% acetonitrile) and solvent B (acetonitrile containing 5% water). A flow rate of 4 mL min−1 with a linear gradient of 0−30% B was applied over 20 or 30 min. The ODN was quantified using UV−vis spectroscopy. The sequences were confirmed by MALDI-TOF mass spectrometry analysis. The trityl group was removed by adding a silver nitrate solution (150 μL, 50 mM) to the dry ODN sample for 30 min. A cloudy white suspension was observed. Dithiothreitol (DTT; 200 μL, 10 mg/mL) was added and allowed to react for 5 min. A yellow precipitate formed and was removed by centrifugation. The excess DTT was removed on a NAP column with water as eluent. The ODN was used immediately. Spherical Gold DNA Conjugates. The gold nanoparticle solution (3 mL) was placed in a glass sample vial. The 5′ sulfhydryl ODN (0.75 OD) was added and the sample agitated overnight on an orbital shaker. Sodium phosphate buffer (100 mM, pH 7.4) and sodium dodecyl sulfate (SDS, 1%, w/v) were added to give final concentrations of 10 mM and 0.01%, respectively. The particles were shaken for 30 min. Aliquots of NaCl (2 M) were added, increasing the salt concentration by 0.05 M increments over 48 h, until a final concentration of 0.3 M was achieved. The sample was left to equilibrate overnight. The particles were pelleted by centrifugation. The supernatant was removed and the pellet redispersed. This was repeated three times to remove any unconjugated ODN. Oligreen Assay. A 250 μL aliquot of each gold nanoparticle-ODN conjugate was taken and treated with dithiothreitol (DTT; 250 μL, 0.2M). The sample was placed at 40 °C for 30 min. The samples were centrifuged to pellet the particle aggregate and a portion of the supernatant removed for analysis. Oligreen reagent (diluted 200 fold) was added to the samples and the fluorecence measured with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. A standard curve was prepared for each ODN with known ODN concentrations. AuAg Nanobox-DNA Conjugates. The AuAg nanobox solution (3 mL) was placed in a glass sample vial. The 5′sulfhydryl ODN (1.5 OD) was added and the sample agitated overnight on an orbital shaker. Sodium phosphate buffer (100 mM, pH 7.4) and sodium dodecyl sulfate (SDS, 1%, w/v) were added to give final concentrations of 10 mM and 0.01%, respectively. The particles were shaken for 30 min. Aliquots of NaCl (2 M) were added, increasing the salt concentration by 0.025 M increments over 48 h, until a final concentration of 0.1 M was achieved. The sample was left to equilibrate overnight. The particles were pelleted by centrifugation. The supernatant was removed and the pellet redispersed. This was repeated three times to remove any unconjugated ODN. Analysis of the supernatant showed that, of the 7.7 nmoles of added ODN, 1.2 nmoles were conjugated to the nanoboxes. UV−vis Spectroscopy. A Cary Varian 50 Scan spectrophotometer and a Perkin-Elmer Lambda 1050 UV/vis/NIR spectrophotometer were used for all UV−vis measurements. Thermal denaturation experiments were performed using a
of our knowledge nanoboxes have not previously been functionalized with ODNs. Shaped particle systems offer the potential of generating interesting assemblies and arrays as well as possible enhanced detection capabilities. As mentioned above, silver nanoparticles have proven much more challenging to functionalize with ODNs due to the ease with which they oxidize.36 For future applications we need to ensure that the particle morphology and properties are preserved. The use of gold coated materials offers enhanced stability and the prepared nanostructures can offer improved properties. In this paper we further characterize the triangular AuAg nanoboxes derived from TSNPs and compare the properties of these nanoboxes functionalized with ODNs to those prepared from spherical gold particles.
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EXPERIMENTAL METHODS Materials. Chemical reagents were purchased from Sigma Aldrich and Fluka excluding the DNA synthesis reagents, which were obtained from Glen Research, and the Quant-iT OliGreen from Invitrogen (Biosciences). All reagents and solvents were used as received unless otherwise indicated. For the nanoparticle preparations distilled water passed through a Millipore Synergy 185 unit was further distilled before use. All glassware for the nanoparticle work was treated with aqua regia and rinsed thoroughly with distilled water before use. Ag Nanoplates and AuAg Nanoboxes Preparation. Triangular silver nanoplates (TSNPs) were prepared using a previously reported method involving a two step seed mediated procedure.25 The seed particles were prepared by stirring a mixture of water (4.5 mL), poly(sodium styrenesulphonate) (PSSS; 0.25 mL, 500 mg/L; 1000 kDa), trisodium citrate (0.5 mL, 25 mM), and sodium borohydride (0.3 mL, 10 mM, freshly prepared) in a beaker. An aqueous silver nitrate solution (5 mL, 0.5 mM) was then added via syringe pump at a rate of 2 mL/min. The TSNPs were prepared by mixing seeds (100 μL) with water (5 mL) and ascorbic acid (75 μL, 10 mM) with stirring in a beaker. An aqueous silver nitrate solution (3 mL, 0.5 mM) was then added via syringe pump at a rate of 1 mL/ min. These TSNPs were converted to the AuAg nanoboxes as follows.39 An excess of ascorbic acid (825 μL, 10 mM) was added to a stirred solution of Ag nanoplate sol and gold(III) chloride trihydrate solution (10 mL, 0.5 mM) was added at a rate of 1 mL/min via syringe pump. The particles were used immediately without the addition of any stabilizers. Spherical Gold Nanoparticles.50,51 HAuCl4 (500 mL, 1 mM) was placed in a round-bottom flask (1 L) fitted with a condenser and brought to the boil with vigorous stirring. Trisodium citrate (50 mL, 38.8 mM) was added. The mixture was refluxed for 20 min. The solution turned from pale yellow to deep red. The solution was allowed to cool to room temperature and was filtered (0.2 μm Omnipore filter). The resulting solution of colloidal particles was characterized by an extinction maximum at 519 nm. TEM analysis showed the particle size to be 13 nm (SI). The particles were stored in the dark at 4 °C before use. Synthesis of ODNs. Automated DNA synthesis was carried out on an Applied Biosystems ABI 3400 DNA synthesizer using standard β-cyanoethyl nucleoside phosphoramidite chemistry on a 1 μmole scale. The ODNs were 5′-modified with a trityl protected sulfhydryl group by incorporating the commercially available S-trityl-6-mercaptohexyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research) during the last 670
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PerkinElmer Lambda 35 UV−vis spectrophotometer equipped with a PTP1 peltier temperature controller. Thermal Denaturation of Nanoparticle ODN Assemblies. Equimolar amounts of the complementary nanoparticle ODN conjugates in 0.3 M PBS were mixed and allowed to hybridize and precipitate overnight. Samples were heated at a rate of 0.5 °C/min, from 25 to 80 °C, taking a reading every 1 °C. The samples were monitored at 260 nm or at the λmax for the nanoparticle solutions (520 nm for Au and 760 nm for AuAg nanoboxes). Thermal Denaturation of Free Duplex ODN. A solution of the two complementary sequences [8 μM] was prepared in 0.3 M NaCl, 10 mM sodium phosphate buffer pH 7. The sample was annealed for 5 min at 95 °C and allowed to cool gradually to room temperature and placed at 4 °C overnight to ensure hybridization. The change in extinction of the hybridized DNA duplex was monitored at 260 nm by incrementally heating the sample from 5 to 80 °C. Transmission Electron Microscopy (TEM). Samples were prepared for TEM by deposition and drying of a drop of an aqueous solution on to a 300 mesh carbon grid. TEM measurements were carried out with a JEOL JEM-2100 LaB6 operating at 200 keV. EDS and HAADF-STEM. High angle annular dark fieldscanning transmission electron microscopy (HAADF-STEM) images were taken of samples that had been prepared for TEM. The HAADF-STEM imaging was carried out with a Titan 80300 TEM operating at 300 kV with Z-contrast imaging conditions that avoided the collection of any electrons that arise due to Bragg scattering. EDS maps were obtained with and EDAX r-TEM detector with a 30 mm2 detector crystal. Dynamic Light Scattering. Particle sizing measurements were carried out using a Malvern Instruments Zetasizer Nano ZS. Measurements were performed using the 173° backscatter setup. Temperature experiments were carried out in a quartz cuvette using a temperature trend method. The sample was heated from 20 to 65 °C at 1 °C interval.
Figure 1. (A and B) EDS maps of flat-lying triangular AuAg nanoboxes produced from X-rays corresponding to the Au-M line (A) and Ag-L line (B) illustrating the distributions of Au and Ag respectively. The white boxes I, II, and III indicate areas from which EDS line profiles were taken, integrated across the width of the box, in the directions indicated by the white arrows. (C) STEM-HAADF image of triangular AuAg nanoboxes before EDS mapping. (D) STEM-HAADF image of same AuAg nanoboxes as in panel C but after exposure to electron beam during EDS mapping.
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RESULTS AND DISCUSSION Formation and Properties of Au−Ag Nanostructures. Hollow triangular AuAg nanoboxes were prepared from triangular silver nanoplates (TSNPs) as previously reported.39 HAuCl4 was added to the TSNPs in the presence of excess reducing agent, ascorbic acid, taking advantage of the galvanic displacement reaction to yield triangular nanobox structures. In this earlier article we provided conclusive evidence for the hollow morphology of the nanoboxes. Although it was shown that the nanoboxes consisted of a AuAg alloy, further analysis of the nanoboxes is needed to obtain a clearer picture of the distribution of Au and Ag within the triangular nanoboxes. To address this we have carried out energy dispersive X-ray spectroscopy (EDS) mapping of flat-lying AuAg nanoboxes (Figure 1A,B). From these EDS maps it is clear that the distributions of Au and Ag indicate that the interior of the particle is indeed a cavity and not a core of Ag and also that the Au and Ag are for the most part well alloyed. The EDS maps however, show that the distributions of Au and Ag are not perfectly matched, indicating that the Au and Ag do not form a homogeneous alloy throughout. To conduct a closer examination of this phenomenon, we obtained wide line profiles across the edges of flat-lying nanoboxes, in the three areas marked in Figure 1A,B. The profiles have been normalized and averaged (Figure 2; the individual profiles SI
Figure 2. Averaged EDS line profiles for Au and Ag for each of the areas I, II, and III indicated in Figure 1A,B illustrating the distribution of Au and Ag across the edge wall of the nanoboxes. The signal is plotted as a function of distance in the direction indicated by the white arrows. The shaded regions are overlaid onto a schematic of the edge wall of a nanobox to illustrate the variation in the distribution of Au and Ag. Note that the ratio of signal intensities is not the absolute molar ratio.
Figure 1S). High angle annular dark field scanning transmission electron microscopy (HAADF-STEM) images before and after the EDS mapping (Figure 1C,D, respectively) were obtained, 671
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which showed that there was minimal damage to the nanoboxes during the scan. From the profiles in Figure 2, it is clear that the edge wall of the nanoboxes can be divided into three sections. That at the edge wall closest to the interior cavity and that at the outermost section are both Au-rich, whereas in between these two areas there is a region that is richer in Ag. The higher amount of gold in the innermost section of the edge wall is the result of epitaxial deposition of a thin layer of Au at the high energy edges of silver nanoplates, which we reported in detail previously.38 The higher amount of gold in the outermost section arises from a thin layer of gold that has been deposited around the whole nanobox. In our previous article,39 we reported that fully enclosed nanoboxes can be formed from a typical batch of Ag nanoplates at a Ag:Au of 1:2.7 while the nanobox samples produced here have a stoichiometry of 1:3.3, i.e., proportionately 20% more Au. All of the Ag from the template Ag nanoplates has been oxidized and then incorporated into the alloyed walls of the nanoboxes by co reduction of Ag+ and AuCl4¯ by ascorbic acid. This means that any further reduction of AuCl4¯ will result in the deposition of a layer of Au around the nanoboxes. It is this external layer of Au that gives rise to the enrichment of the outermost section of the edge wall with Au, as detected in the EDS line profiles. The HAADF-STEM images may also be consistent with the extra gold forming a rounded raised surface at the edge of the nanoplate. This layer of gold on the outside is very important for the application of nanoboxes in biological systems. Gold is an inert substrate yet is an ideal platform for the immobilization of biomolecules due to the ability of thiol functional groups to bind strongly to gold. This outer coating of gold should favor the successful functionalization of triangular AuAg nanoboxes with thiol-modified oligonucleotides. We now have a clearer picture of the structure of the nanoboxes and how they form. A schematic diagram illustrating the different stages of an updated and more complete growth model for the formation of triangular nanoboxes is shown in Figure 3. ODN Functionalization of Spherical Gold Nanoparticles. Gold surfaces are readily functionalized with thiols and this has been used extensively to derivatize gold nanoparticles with ODNs.40−44,52 To begin our investigation, spherical gold nanoparticles were prepared following a previously reported procedure53 and functionalized with 5′thiol modified ODN sequences 1 and 2 (Figure 4). The decameric adenosyl spacer was incorporated to overcome the nonspecific adsorption of the nucleobases with the metal surface, allowing for increased ODN loading.54,55 The spacer also aids in distancing the ODN from the nanoparticle surface ensuring efficient hybridization. The gold nanoparticle samples were gradually brought to a final salt concentration of 300 mM NaCl over 48 h and the excess ODN removed by centrifugation. A 5 nm red shift was observed in the LSPR band from 519 to 524 nm (SI, Figure 2S). An Oligreen assay was used to estimate the loading of the single-stranded ODN on each nanoparticle.56 Sequence 1 was found to have a lower value of 71 strands/particle and sequence 2 had a slightly higher value of 117 strands/particle. These values are comparable with the literature reported value of 35 pmol/ cm2 (112 strands for a 13 nm particle). Our results indicate that the composition of the sequence has an effect on the loading of the particles. Both strands contain an identical stretch of adenines, which as noted previously should aid high loading. Therefore it may be proposed that the lower loading of
Figure 3. Schematic illustrating formation of a triangular AuAg nanobox from a silver nanoplate template.
Figure 4. Schematic showing the cross-section of a thiolated-ODN functionalized hollow triangular AuAg nanobox.
sequence 1 might be a result of the attraction of the G nucleobase for the nanoparticle surface.54 When Au conjugates with complementary ODNs 1 and 2 were mixed, a color change from red to purple was observed. To compare the relative stability of this nanoparticle assembly to that of the duplex formed from 1 and 2, their melting temperatures were obtained by thermal denaturation experiments (SI, Figure 3S). The free duplex was determined to have a melting temperature of 36 °C whereas the nanoparticle assembly denatured at a higher temperature of 45 °C with a significantly sharper transition. These observations are consistent with previous reports,40,57,58 with the increased temperature and sharpness being attributed to the cooperative melting effect of densely functionalized nanoparticles. 672
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ODN Functionalization of AuAg Nanoboxes. The functionalization of triangular AuAg nanoboxes with ODNs was achieved by adopting a similar method to that used for the spherical gold particles with identical ODN sequences. However a modified procedure was required as the “as prepared” nanoboxes undergo irreversible aggregation when the salt concentration exceeds 30 mM, even though the particles were observed to have a similar zeta potential (−47.3 mV) to that of the spherical gold nanoparticles (−31.4 mV) before conjugation. This problem was solved by adopting a very slow salt aging process and adding a greater excess of DNA. The AuAg nanoboxes were used immediately after preparation without the addition of any stabilizers. The ODNs were added directly to the AuAg nanobox solution (Figure 4), and the solution was agitated overnight. The salt concentration was slowly increased over 48 h to a final concentration of 100 mM NaCl, 10 mM sodium phosphate, and 0.01% SDS. The addition of salt has been shown to result in increased ODN loading probably due to the increased electrostatic screening between ODN strands.59 Interestingly sequence 1, which has the lower loading value for the spherical particles, was found to be the most problematic. Larger, nanoplate structures are more like planar plates, and the ODN packing density on the {111} is expected to be less, resulting in the particles being more susceptible to irreversible aggregation.44 The excess unconjugated ODN was removed by multiple washing steps, centrifuging, and resuspending the nanoparticles in 100 mM NaCl, 10 mM sodium phosphate, and 0.01% SDS. From an analysis of the supernatant (see the Experimental Methods), it was estimated that ca. 1.2 nmol of ODN were attached to the nanoplates yielding an estimated surface coverage of approximately 60 pmol/cm2. Analysis of the sample by UV−vis spectroscopy showed the main LSPR band to have red-shifted by 18 nm (from 744 to 762 nm) on ODN functionalization (Figure 5). The refractive
a dampening in the LSPR band was observed on formation of the assembly. The sample precipitated when left overnight. To determine the reversible nature of the assembly, the aggregate was redispersed by agitation and the absorbance at 760 nm (the main LSPR band of the AuAg nanoboxes) of the hybridized conjugate was measured as a function of temperature (Figure 7A). A sharp increase in absorption occurred at 37 °C. (It may be noted that a sample of citrate stabilized AuAg nanoboxes showed no phase transition with increasing temperature.) The increase in extinction observed at the LSPR band of the particles (760 nm) indicated that the transition was as a result of the dehybridization of the AuAg nanobox-ODN assembly, with the recovery of the initial spectrum of the particles obtained. Significantly this melting transition was found to be reproducible, as the repeat melting of the same sample carried out over a week-long period gave a similar transition each time (SI, Figure 4S). This provides strong evidence that the AuAg nanobox-ODN conjugates do not degrade (e.g., by dissociation of the ODNs from the surface during repeated heating/cooling cycles). The melting curve obtained for the nanoboxes was significantly sharper than that of the free duplex but at a similar temperature (Figure 7A). The sharpness results from the cooperative melting of the multiple ODN strands. Previous reports with ODN functionalized triangular nanoplates have resulted in an increase in the Tm compared with spherical particles, and this has been attributed to the face to face assembly with the cooperative effect of the ODN strands being enhanced.43,44 There are a few possibilities to account for this lower melting transition. The ODN packing on specific facets could be different, as previously observed by Mirkin and coworkers.44 If the basal plane is not densely functionalized then the ODN conformation could be such that the ODNs are not readily available for hybridization. The side facets have a higher degree of curvature and are considered to be more reactive, and thus are more likely to be densely functionalized and the hybridization more likely to occur.61 The melting transition of the hybridized AuAg nanobox-ODN systems was observed to occur at higher melting temperatures with increasing NaCl concentration. Thus the melting temperature increases from 37 to 58 °C when the NaCl concentration is raised from 0.3 to 0.6 M (SI, Figure 3S). Dynamic light scattering (DLS) was also used (Figure 7B) to monitor the disassembly. The aggregate size was observed to decrease with increasing temperature (by contrast a citrate stabilized AuAg nanobox solution showed no change in particle size when heating). Transmission electron microscopy analysis of the hybridized AuAg nanobox-ODN conjugates was also carried out (Figure 8). The samples, with identical concentrations, were dropped under the same conditions to eliminate the possibility that the observed result was due to the drying of the sample on the TEM grid. In the absence of the complementary sequence no aggregation occurred (Figure 8A), whereas by contrast, in the presence of the complementary conjugate, an aggregate of the AuAg nanobox assembly was observed (Figure 8B). Both the DLS and TEM of the assembled aggregate showed a smaller size in comparison with the spherical gold nanoparticles, although the individual nanoboxes are substantially larger and this may also explain the lower melting temperature observed. Assembly of Spherical Au-ODN and AuAg-ODN. Further evidence for the successful ODN functionalization of these AuAg nanoboxes was obtained by assembling them with
Figure 5. UV−vis spectra of Ag nanoplates (black circles), AuAg nanoboxes (red squares) and AuAg nanobox-ODN conjugates (blue triangles) prepared using 5′-thiolated ODN.
index sensitivity of these AuAg nanoboxes is greater than the analogous spherical gold and silver nanoparticles and accounts for the larger shift in the LSPR band upon functionalization.60 Equimolar amounts (quantified by UV−vis spectroscopy) of the AuAg nanobox-ODN conjugates functionalized with the complementary sequences 1 and 2 were mixed. No color change was observed until the salt concentration was increased to 300 mM NaCl, when a transition from dark blue to gray was observed by the naked eye (Figure 6A) and by UV−vis spectroscopy (Figure 6B). A red shift from 760 to 820 nm and 673
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Figure 6. (A) Photograph of AuAg nanobox-ODN sample before (left) and after (right) hybridization with a complementary conjugate. The sample turned from dark blue to gray. (B) UV−vis spectra of AuAg nanobox-ODN before (blue circles) and after (gray triangles) hybridization with the complementary AuAg nanobox-ODN conjugates. The LSPR band red shifts and dampens.
Figure 7. (A) Thermal denaturation curves for the free duplex ODN (red squares, 0.3 M NaCl, 8 μM, monitored at 260 nm) and the hybridized AuAg nanobox-ODN (black circles, 0.3 M NaCl, monitored at 760 nm). The citrate stabilized AuAg nanoboxes (blue triangles, monitored at 760 nm) showed no effect. (B) Dynamic light scattering analysis of complementary AuAg nanobox-ODN conjugate assembly (black squares) in 0.3 M NaCl as a function of temperature showing the particle size with increasing temperature. The data shown is an average of three measurements. A citrate stabilized AuAg nanobox sol (red circles) was monitored as a control.
TEM analysis was carried out to observe these assemblies (Figure 10, SI Figure 5S). This shows a denser clustering of the Au particles at the edges of the nanoboxes, rather than on the {111} faces. Although the possibility of this clustering occurring due to drying of the sample cannot be excluded, we believe it is more consistent with more ready hybridization of the ODNs at the edges. It is expected that these facets are more densely functionalized due to their higher reactivity compared to the {111} face. Curvature is known to have an effect on the particle loading, and the loading on planar surfaces is proposed to be reduced due to the repulsion of the negative backbones of the ODNs.61,62 This might therefore account for this observation. The ODN on the {111} face may also be orientated in a way that is not accessible for hybridization. These observations could help account and support the lower Tm obtained for the nanobox−nanobox assembly system.
Figure 8. TEM analysis of AuAg nanobox-ODN conjugates: (A) noncomplementary conjugates and (B) complementary conjugates. The samples were dropped in the same concentration, under the same conditions to exclude that the aggregation observed was as the result of a drying effect on the TEM grid.
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spherical gold nanoparticles functionalized with the complementary ODN sequence. The Au-ODN-2 (excess) was added to the complementary AuAg nanobox-ODN-1 system. Both the LSPR band for the Au and the AuAg nanoboxes were observed to red shift and dampen (Figure 9A). No change in the LSPR bands was observed on mixing noncomplementary conjugates. The reversible nature of the assembly was observed by monitoring the thermal denaturation of the system at 760 and 520 nm. A sharp phase transition with a melting temperature of 45.5 °C was observed (Figure 9B).
CONCLUSIONS
As has been previously reported, TSNPs have many favorable properties (especially their tunable plasmon bands) for biosensor and related applications. However their poor stability in salt solutions and their propensity to oxidize can be a limiting factor. By treating the samples with gold (and forming nanobox structures) we demonstrate here that robust particles are produced, which are an excellent platform for ODN conjugation, where high chloride concentrations are used. 674
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Figure 9. (A) UV−vis spectra for the assembly of complementary Au-ODN and AuAg nanobox-ODN conjugates (red triangles) and the noncomplementary conjugates (black squares) in 0.3 M PBS. (B) Normalized melting curve obtained for the thermal denaturation from the assembly of complementary Au-ODN and AuAg nanobox-ODN conjugates in 0.3 M PBS.
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ACKNOWLEDGMENTS We thank SFI (Grant No. 06/RF/CHP036), COST D35, and IRCSET for financial support; Mr, Neal Leddy at the Centre for Microscopy and Analysis (CMA), TCD, for help with the TEM measurements; and Dr. Markus Boese at the Advanced Microscopy Laboratory (AML) in CRANN, for carrying out the HAADF- STEM and EDS mapping. The Nanobio program Université Joseph Fourier, Grenoble is acknowledged for the DNA synthesis and purification facilities.
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Figure 10. TEM images of the assembly from complementary AuDNA and AuAg nanobox-DNA conjugates.
It is interesting to note that when the nanoboxes with complementary ODNs are brought together the Tm is significantly less than that observed with pure gold or silver triangular nanoplates.44,45 This may possibly be a consequence of the nanoboxes’ morphology, in that ODNs bound to the {111} faces are not able to approach sufficiently closely to facilitate hybridization. This may be due to the gold coating at the edge of the particles being raised above the level of this triangular face. It may also be noted that the hybridizing section of our ODN is relatively short (9-mer). The hybridization of the nanoboxes with the functionalized gold nanoparticles may be caused by preferred binding at the edges. As pointed out above, this might be due to the rounded nature of the edges and perhaps to a somewhat different elemental composition (the edges being expected to be richer in gold). Future studies should be directed to using these particles for biodiagnostic applications exploiting their stability and excellent properties as well as additionally using encapsulation and targeting with nucleic acids.
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AUTHOR INFORMATION
REFERENCES
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S Supporting Information *
Supporting figures. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author
*Fax: +353 1 6712826. Tel: +353 1 8961947. E-mail: jmkelly@ tcd.ie. Notes
The authors declare no competing financial interest. 675
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